All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
The gram-positive spore-forming bacterium Bacillus anthracis (B. anthracis) is the causative agent of anthrax, a rare fatal disease which is initiated, in its most severe form, by inhalation of infected spores. Due to the severity of the disease, the ease of respiratory infection and the everlasting resistance of the spores to unfavorable environmental conditions, B. anthracis is considered a potential biological warfare agent (for review, see Mock and Fouet, 2001), and in recent years the need for novel reliable diagnostic approaches, improved vaccination strategies, novel therapeutic targets and better understanding of the pathogenesis, have been widely acknowledged.
Inhaled B. anthracis spores are taken up by alveolar macrophages, germinate into vegetative bacilli which eventually invade the blood stream where they multiply massively and secrete toxins and virulence factors. Anthrax is toxinogenic in the sense that the bacterial binary exotoxin is necessary for the onset of the disease (Lacy and Collier, 2002), yet other factors may be require for the colonization and expansion of the bacteria in the host (for example Cendrowski et al., 2004; Gat et al., 2005). The toxin is composed of three proteins: protective antigen (PA, which mediates binding to the receptor on target cells and internalization of the toxin), lethal factor (LF, a zinc protease targeting MAP kinase) and edema factor (EF, a calmodulin dependent adenylate cyclase; Leppla, 1999). The genes encoding for the three exotoxin components are located on the native pXO1 virulence plasmid. Genes encoding for functions involved in the synthesis of a polyglutamyl immunologically-inert capsule that protects bacteria from phagocytosis are located on a second native virulence plasmid, pXO2 (Leppla, 1999).
In humans, the initial symptoms of anthrax inhalation are nonspecific, reminiscent of influenza-like, upper respiratory track infections. The second stage is characterized by high fever, respiratory failure, dyspnea and shock. Unless promptly treated, death occurs within 24 hours of the onset of the second stage preceded by massive bacteremia (Dixon et al., 1999; Shafazand et al., 2008; Stern et al., 2008). The mandatory treatment for anthrax is based on administration of antibiotics (Stern et al., 2008; Bryskier et al., 2002), yet studies of the disease in animal models have clearly established that the time of antibiotic administration post infection is crucial for the effectiveness of the treatment, strongly supporting the importance of rapid diagnosis (Altboum et al., 2002). As of today, due to the severity of the disease and its rapid progression, treatment is administrated in each confirmed contact with contaminated areas (for review, see Stern et al. 2008).
Early accurate diagnosis of anthrax is complicated by the rarity of the disease and the nonspecific nature of the symptoms. Microbiologic identification of anthrax is based on the non-hemolytic nature of the typically white/gray colonies and the rod morphology of the gram positive non-motile bacilli detected in clinical samples or blood cultures (Turnbull, 1999; Edwards et al., 2006; Shafazand et al., 1999). Immuno-fluorescence and immunohystochemistry targeted to bacterial proteins are not routinely conducted. Later in the course of the disease, seroconversion to the various components of the toxin may serve only as a retrospect confirmation of primordial exposure. With the advent of genetic methodologies, B. anthracis is specifically and accurately detected in cultures inoculated with clinical and forensic samples, by PCR usually designed to amplify genes located on the native virulence plasmids (Edwards et al., 2006). The use of PA as a disease biomarker has been suggested owing to the presence of this protein in detectable amounts in the circulation of infected animals (Kobiler et al., 2006; Rossi et al., 2008). EF and LF can be detected in the circulation only at very late stages of the disease, aborting their use as disease biomarkers (Edwards et al., 2006).
In recent years, the search for novel disease biomarkers in general and bacterial infection in particular has exploited the approach of the global biological inspection based on functional genomic or proteomic studies (Wu et al., 2008). The present inventors have documented previously such global surveys combined with serological study of B. anthracis for identification of vaccine and diagnostic marker candidates among exposed (secreted or membranal) proteins (Ariel et al., 2002, 2003; Chitlaru et al., 2004, 2006, 2007; Gat et al., 2006).
Therefore, it is an object of the present invention to provide a method for the diagnosis of anthrax, through the specific and rapid detection of biomarkers described by the inventors. In particular, the inventors describe herein a method for diagnosing B. anthracis infection through the detection of three secreted proteins, namely the products of locus BA3660, BA1952 and BA0796.
As will be shown by the specification and the following Examples, the diagnostic method of the invention is particularly suitable for detection and monitoring of B. anthracis infection. Moreover, the sensitive diagnostic method of the invention may be applied for early detection and diagnosis of anthrax, as well as for monitoring efficiency of therapeutic treatments and delayed confirmation of B. anthracis infection.
These and other uses and objects of the invention will become apparent as the description proceeds.